Progressive hypertrophy and heart failure in beta1-adrenergic receptor transgenic mice

Abstract

Stimulation of cardiac beta1-adrenergic receptors is the main mechanism that increases heart rate and contractility. Consequently, several pharmacological and gene transfer strategies for the prevention of heart failure aim at improving the function of the cardiac beta-adrenergic receptor system, whereas current clinical treatment favors a reduction of cardiac stimulation. To address this controversy, we have generated mice with heart-specific overexpression of beta1-adrenergic receptors. Their cardiac function was investigated in organ bath experiments as well as in vivo by cardiac catheterization and by time-resolved NMR imaging. The transgenic mice had increased cardiac contractility at a young age but also developed marked myocyte hypertrophy (3.5-fold increase in myocyte area). This increase was followed by progressive heart failure with functional and histological deficits typical for humans with heart failure. Contractility was reduced by approximately 50% in 35-week-old mice, and ejection fraction was reduced down to a minimum of approximately 20%. We conclude that overexpression of beta1-adrenergic receptors in the heart may lead to a short-lived improvement of cardiac function, but that increased beta1-adrenergic receptor signalling is ultimately detrimental.

Figure 1

Generation and identification of mice transgenic for the β₁-adrenergic receptor. (A) Transgenic vector. The complete intergenic region between the β- and α-myosin heavy chain genes (α-myosin heavy chain promoter) was ligated 5′ to the coding region of the human β₁-adrenergic receptor. At the 3′ end of the receptor cDNA, the intron and poly(A)-containing sites of the simian virus 40 T antigen were added. β, α1, α2, and α3 denote exons of the β- and α-myosin heavy chain genes. The positions of the PCR primers used to detect the transgene (B) are indicated. (B) Detection of the transgene in animals of the lines β₁TG7 and β₁TG4 by PCR with the primers indicated in A. The lanes at the right side contained 1 and 10 pg of the transgene vector in the PCR as standards and a negative control (C) without genomic DNA. αMHC, α-myosin heavy chain.

Figure 2

Expression and function of β₁-adrenergic receptors in transgenic animals. (A) Detection of β-adrenergic receptors in left ventricular myocardial membranes prepared from wild-type mice (WT) and mice from the two transgenic lines β₁TG7 and β₁TG4. β-Adrenergic receptors were quantified by saturation with the radioligand [¹²⁵I]cyanopindolol. The data shown are means ± SEM of four experiments. (B) Frequency responses in organ bath experiments. Right atria from 8- to 12-week-old wild-type mice (▫) and mice from the two transgenic lines β₁TG7 (▪) and β₁TG4 (●) were mounted in an organ bath. Spontaneous contraction frequencies were recorded under basal conditions and in the presence of increasing concentrations of (−)isoproterenol. EC₅₀ values were 1.5 ± 0.2 nM for wild-type mice, 1.1 ± 0.1 nM for β₁TG7, and 0.4 ± 0.05 nM for β₁TG4, and maximal effects were 233 ± 9, 253 ± 4, and 270 ± 8 beats per min, respectively. The data shown are means ± SEM of nine (WT), six (β₁TG7), and four (β₁TG4) atria.

Figure 3

Histological alterations in transgenic hearts. (A and B) Hematoxylin/eosin-stained 5-μm sections of paraffin-embedded left ventricular myocardium from wild-type (A) and β₁TG4 mice (B). (C) Morphometrical analysis of myocyte cross-sectional areas. Myocyte cross-sectional areas were determined from hematoxylin/eosin stained sections from β₁TG4 (●), β₁TG7 (▪), and wild-type left ventricles. Data are expressed as percentage of the respective wild-type value and represent means ± SEM of several sections from three animals at each age group.

Figure 4

In vivo determination of left ventricular contractility in β₁TG4 and wild-type mice. Left ventricular pressures in anaesthetized mice were measured with a Millar 1.8 French catheter. (A) Left ventricular maximal contractility (dp/dt_max) was measured at different ages in wild-type (▫) and β₁TG4 (●) mice. Data are from four mice for each age group. The Inset shows an original pressure tracing. (B) Effects of β-adrenergic receptor blockade by 1.5 μg of propranolol per g of body weight on dp/dt_max in wild-type and β₁TG4 transgenic animals at 16 weeks of age. mmHg, millimeters of mercury.

Figure 5

Detection of heart failure in β₁TG4 transgenic mice by NMR imaging. ECG-triggered series of NMR images of 35-week-old transgenic and wild-type (WT) mice were recorded with a 7.05 T BIOSPEC 70/20 scanner (20). The Inset shows an image in maximal diastole. LV, left ventricle; RV, right ventricle; CW, chest wall; Li, liver; BV, blood vessel (of portal circulation). Ejection fractions were calculated from images in maximal diastole and systole (20).